Interleukin 2 (IL-2) is a pluripotent cytokine produced primarily by activated CD4+ T cells, which plays a crucial role in producing a normal immune response. IL-2 promotes proliferation and expansion of activated T lymphocytes, potentiates B cell growth, and activates monocytes and natural killer cells. It was by virtue of these activities that IL-2 was tested and is used as an approved treatment of cancer (aldesleukin, Proleukin®).
In eukaryotic cells human IL-2 is synthesized as a precursor polypeptide of 153 amino acids, from which 20 amino acids are removed to generate mature secreted IL-2 (Taniguchi 1983). Recombinant human IL-2 has been produced in E. coli (Rosenberg 1984), in insect cells (Smith 1985) and in mammalian COS cells (Taniguchi 1983).
IL-2 works by interacting with three different receptors: the interleukin 2 receptor alpha (IL-2Rα; CD25), the interleukin 2 receptor beta (IL-2Rβ; CD122), and the interleukin 2 receptor gamma (IL-2Rγ; CD132; common gamma chain). The first receptor to be identified was the IL-2Rα, which is a 55 kD polypeptide (p55) that appears upon T cell activation and was originally called Tac (for T activation) antigen. The IL-2Rα binds IL-2 with a Kd of approximately 10−8 M, and is also known as the “low affinity” IL-2 receptor. Binding of IL-2 to cells expressing only the IL-2Rα does not lead to any detectable biologic response.
The IL-2Rβ is a member of the type I cytokine receptor family characterized by the two cysteine/WSXWS motif. The IL-2Rβ is expressed coordinately with the IL-2Rγ. The IL-2Rγ, a 64 kD polypeptide, is also known as the common γ chain because it is shared among a number of cytokine receptors, including the receptor for interleukin-4 and interleukin-7. The IL-2Rβγ is the same signaling receptor complex that can bind to IL-15.
Most cells, for example, resting T cells are insensitive to IL-2 since they only express the IL-2Rβ and the IL-2Rγ. Upon antigen receptor-mediated T cell activation, the IL-2Rα is rapidly expressed. Once the IL-2Rα binds IL-2, it then sequentially engages the IL-2Rβ and the IL-2Rγ (FIG. 1). IL-2 binding by the IL-2Rαβγ complex results in signal transduction through a Jak/STAT signaling pathway and IL-2 mediated growth stimulation.
So far, only limited structure/function analysis of human IL-2 has occurred, although analysis of mouse IL-2 has been extensive (Zurawski, S. M. and Zurawski, (1989) Embo J 8: 2583-90; Zurawski, S. M, et. al., (1990) Embo J 9: 3899-905; Zurawski, G. (1991). Trends Biotechnol 9: 250-7; Zurawski, S. M. and Zurawski, G. (1992) Embo J 11: 3905-10. Zurawski, et. al., EMBO J, 12 5113-5119 (1993)). Some human IL-2 muteins have been examined for their activity on human PHA blasts (Xu, et. al., Eur. Cytokine Netw, 6, 237-244 (1995)). Other examples of human IL-2 muteins are provided by Buchli and Ciardelli, Arch. Biochem. Biophys, 307(2): 411-415, (1993), Collins, L., et al., PNAS USA 85:7709-7713 (1988), and U.S. Pat. No. 5,696,234 (Zurawski et al.).
The use of IL-2 as an antineoplastic agent has been limited by the serious toxicities that accompany the doses necessary for a tumor response. The major side effect of IL-2 therapy is vascular leak syndrome (VLS), which leads to the accumulation of intravascular fluid in the lungs and liver resulting in pulmonary edema and liver damage. Until recently it was believed that VLS was caused by the release of proinflammatory cytokines from IL-2 activated NK cells. However, a recent report points to the direct binding of IL-2 to lung endothelial cells, as a purported cause of VLS. (Krieg et al., PNAS USA 107(26)11906-11911 (2010). In principle, an IL-2 variant with high affinity for IL-2Rβ, whose activity was not dependent on CD25 expression could have improved clinical utility and reduced toxicity.
One IL-2 mutein of clinical interest is BAY 50-4798, which differs from wild-type IL-2 by the substitution of arginine for asparagine at position 88 (R88N) (Steppan et al. (2006) J. Interferon and Cytokine Res., 26(3): 171-. This modification allegedly results in an IL-2 mutein with relatively reduced binding to the IL-2Rβγ, and thought to possess lower toxicity relative to wild type IL-2. However, a clinical study found that patients receiving BAY 50-4798 experienced a similar degree of IL-2 mediated VLS.
For these reasons, it is clear that IL-2 muteins that exhibit unique properties are needed. Potential uses of such muteins include treating cancer (as a direct and/or adjunct therapy) and immunodeficiency (e.g., HIV and tuberculosis). Other potential uses of IL-2 are derived from its immunostimulatory activity, and include direct treatment of cancer, treating immunodeficiency, such as HIV or human SCID patients; treating infectious disease, such as tuberculosis; its use as an adjuvant in “cancer vaccine” strategies; and for immune system stimulation indications, such as enhancing standard vaccination protocols (e.g., elderly). For example, IL-2 muteins that exhibit reduced VLS would be advantageous.
The present disclosure provides novel IL-2 muteins.